Project Summary/Abstract Metastatic melanoma is a highly aggressive cancer, making it difficult to target via classical cancer therapies. Immunotherapy represents a quickly emerging strategy for targeting aggressive cancers, however a major hurdle is the immunosuppressive environment within the melanoma tumor mediated by Regulatory T cells (Tregs). Current Treg targeting approaches are non-specific and have only transient efficiency, therefore the development of new therapies to control Treg function are critical for immunotherapy. Treg suppressive function is mediated by the transcription factor Forkhead Box P3 (FoxP3), also a specific marker for the Treg cell line. Our preliminary data show that the Ubiquitin-specific peptidase 22 (USP22) is required for proper FoxP3 expression, suggesting a critical role for USP22 in Treg suppressive function and stability. The long term goal of this project is to understand the function of USP22 on Treg suppressive function in the context of cancer. Our lab generated the first conditional knockout mouse model of USP22, and preliminary data show a dramatic decrease of FoxP3 expression upon USP22 deletion as well as chronic activation of CD4+ and CD8+ T cells. Importantly, a deletion of USP22 in all T cells showed no defect in T effector cell activation, indicating USP22 as an ideal therapeutic target for modulating immunosuppression within the tumor microenvironment. Challenging of both WT and mice harboring a USP22 genetic deletion in Tregs (USP22Treg-KO) with EG7 lymphoma showed an increased in anti-tumor response in the USP22Treg-KO background. The function of USP22 on Tregs in the context of melanoma, a more aggressive cancer, has never been studied. Based on the data obtained, the central hypothesis for this study is that USP22 is critical for Treg stabilization and immunosuppression and that genetic USP22 suppression enhances melanoma antitumor immunity in mice. The hypothesis will be addressed in three aims. Aim 1 will focus on studying the role of USP22 on maintaining Treg suppressive function. Aim 2 will determine the specific mechanisms by which USP22 mediates FoxP3 expression. Aim 3 will address the role of USP22 on Treg function in the context of melanoma. The methods that will be used to accomplish the proposed aims will include novel transgenic mouse models, tumor models, as well as biochemistry, molecular, and immunological techniques. The studies will provide fundamental insights to Treg biology and regulation, and the function of USP22 on immune suppression and cancer.